Abstract: The proper folding of proteins is essential for all life, making aberrant protein folding severely problematic. The protein homeostasis (proteostasis) network is a tightly regulated system that ensures that proteins are folded and degraded as necessary. However, misfolded proteins can overwhelm the proteostasis network. Protein misfolding is associated with numerous devastating neurodegenerative and cardiovascular disorders. Protein disaggregases can engage misfolded substrates and unfold them, promoting their return to proper fold and function. Disaggregases may serve as the final defense against collapse of the proteostasis network. Disaggregases play key roles in maintaining cellular health, and can even regulate beneficial amyloids in yeast. Amyloid is an exceptionally stable protein conformation that is implicated in numerous human diseases, and amyloid is considered to be otherwise intractable. I hypothesize that disaggregases play key roles in maintaining cellular health, but are vulnerable when the proteostasis network becomes overwhelmed in disease. Therefore, technologies that modulate disaggregase activity might be therapeutically useful. However, protein disaggregases are the least well characterized branch of the proteostasis network. I envision building a research program focused on developing a more comprehensive understanding of how disaggregases counter misfolding, both in health and under stress. I seek to elucidate how cells maintain proteostasis, how proteostasis fails, and how protein disaggregases might ultimately be applied to prevent or even reverse collapse of proteostasis. To further these goals, we will focus on three main themes over the next five years: (1) we will develop and apply new technologies to study and modulate the substrate-specificity of Hsp104, which regulates beneficial amyloid conformers in yeast. (2) We will characterize newly identified human amyloid disaggregases to better understand their normal roles in maintaining cellular health and how they might fail in disease. (3) We will apply new mass spectrometry techniques to study how protein-remodeling factors select specific substrates to target and how this varies under stress conditions. Our work will elucidate how disaggregases target specific substrates. Additionally, these finely-tuned disaggregases can be used as probes to test the hypothesis that misfolded species are toxic, and that restoration of proteins to their native folds and functions can reverse disease phenotypes. Ultimately we aim to apply the findings from these studies to develop new strategies to treat neurodegenerative disease. This is especially important because, despite intense efforts, there are no therapeutics available to treat protein-misfolding disorders.